Journal of Apicultural Research 49(3): 257-264 (2010) © IBRA 2010

DOI 10.3896/IBRA.1.49.3.05

ORIGINAL RESEARCH ARTICLE

Evaluation of the toxicity of a propolis extract

on Varroa destructor (Acari: Varroidae) and
Apis mellifera (Hymenoptera: Apidae)

Natalia Damiani1,2*, Matías Daniel Maggi1,2, Liesel Brenda Gende1,2, Claudia Faverin3, Martín Javier
Eguaras1,2 and Jorge Augusto Marcangeli1
1
Laboratorio de Artrópodos, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3350,
Mar del Plata 7600, Argentina.
2
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.
3
Grupo Sistemas de Producción, Departamento de Producción Animal, EEA INTA Balcarce, cc 276, Balcarce 7620, Argentina.

Received 15 July 2009, accepted subject to revision 17 March 2010, accepted for publication 15 April 2010.

*Corresponding author: Email: ndamiani@mdp.edu.ar

Summary
The effects of a propolis extract on Varroa destructor and Apis mellifera were evaluated by three different application methods: topical,
spraying and oral. A propolis sample was extracted and its organoleptic and physic-chemically traits characterized. These analyses showed
that it was a typical propolis from the Pampean region in Argentina, with elevated contents of biologically active compounds. Topical
application was carried out by subjecting mites to contact with various propolis concentrations for different periods of time, which resulted in
mortality and narcosis. Acaricidal effects were stronger with increasing concentrations of the propolis extracts. Spraying infested bees with a
10% propolis solution was harmless for bees but killed 78% of mites. Feeding infested bees with propolis extract in sugar syrup was not toxic
to the mites but caused the death of bees treated with the highest concentration. Our results suggest that the propolis extracts from the
Pampean Region could be incorporated into bee colonies by spraying, although the appropriate doses and concentrations to be administered,
and the mechanism of action of the extracts on the mites are still to be elucidated.

Evaluación de la toxicidad de un extracto alcohólico de

propóleos sobre Varroa destructor (Acari: Varroidae)
y Apis mellifera (Hymenoptera: Apidae)
Resumen
Los efectos de un extracto de propóleos sobre Varroa destructor y Apis mellifera fueron evaluados por medio de tres métodos de aplicación
diferentes: tópica, pulverizado y oral. Una muestra de propóleos fue extractada y caracterizada organoléptica y físico-químicamente. Las
características de la muestra resultaron coincidir con las de un propóleos típico de la región pampeana argentina, con un elevado contenido de
compuestos biológicamente activos. La aplicación tópica fue realizada manteniendo a los ácaros en contacto con diferentes concentraciones
de propóleos durante ciertos períodos, resultando en mortalidad y narcosis de los ácaros expuestos. Los efectos acaricidas se incrementaron a
medida que lo hicieron las concentraciones de los extractos de propóleos aplicados. Las abejas infestadas que fueron pulverizadas con una
solución de propóleos al 10% no resultaron afectadas pero el 78% de los ácaros que las parasitaban resultaron muertos. La alimentación de
las abejas con extractos de propóleos en jarabe de azúcar no mostró efectos tóxicos para los ácaros pero causó la muerte de las abejas
tratadas con la concentración más elevada. Nuestros resultados sugieren que los extractos de propóleos de la región pampeana podrían ser
incorporados en las colonias de abejas en forma pulverizada, aunque las dosis y las concentraciones adecuadas a administrar y, el mecanismo
de acción de los extractos sobre los ácaros, aún deben ser elucidados.

Keywords: Varroa destructor; Apis mellifera; propolis extract

258
Introduction
The parasitic mite, Varroa destructor (Anderson and Trueman, 2000),
is considered to be one of the most serious pests of the honey bee,
Apis mellifera, causing great economic losses to the beekeeping
industry (de Jong et al., 1982). Female mites parasitize both adult and as quercetine dihydrate), according to the protocol of IRAM-INTA
brood bees by feeding on their haemolymph, but only reproduce
inside the capped brood cells (Ifantidis, 1983). Efforts to control this
pest have often focused on applying synthetic acaricides. Although
these compounds provide favourable results, the development of
acaricide resistance in V. destructor populations (Milani, 1999) and
the contamination of hive products (Wallner, 1999) indicate that new
treatment strategies that minimize the above hazards should be
developed (Ritter, 1992). Natural acaricides offer a highly desirable
alternative to the synthetic products. Most research has focused on
the use of organic acids and essential oils, because they naturally
occur in bee colonies and possess significant acaricidal activities
(Imdorf et al., 1999; Eguaras et al., 2003). Another natural product
that can be taken into account is propolis.
Propolis is composed of resins collected from plants, which are
masticated by the bees, mixed with their salivary enzymes and
beeswax, and applied to combs and walls of the hive (Burdock, 1998), which had not been treated with any synthetic acaricides. The apiary
thereby insulating and reinforcing the hive as well as giving antiseptic
proprieties to the nest environment (Bankova et al., 2000). The
biological activity of propolis is due to its high resin content, mainly
phenolic compounds (Bankova et al., 1983). Numerous studies have
shown its versatile pharmacological activities: antibacterial; antifungal;
antiviral; anti-inflammatory; antioxidant; antitumour, etc. (Banskota
et al., 2001). Previous tests of the use of propolis as acaricides or
insecticides have, however, been very limited. Garedew et al., (2004)
have shown that topical application of propolis reduced the duration
of the pupal period of the greater wax moth, Galleria mellonella, and
was toxic to its larval instars. More significantly, Garedew et al., (2002) according to each assay (see below).
demonstrated that V. destructor was sensitive to a propolis extract
applied as a topical solution in alcohol. The effect that propolis
extracts could have on the honey bees has not, however, been
investigated. The aim of this work was to evaluate the toxicity of a
propolis alcoholic extract on V. destructor and on A. mellifera by
different means of administration.
Materials and methods
Extraction and analysis of the propolis sample
A sample of raw propolis was obtained from a beekeeper’s apiary
placed in Camet station, Mar del Plata, Buenos Aires province,
Argentina (37º53'S; 57º36'W). The sample was weighed, frozen,
ground with a mortar, and then stored at 4ºC until use. The propolis
was organoleptic and physic-chemically characterized in the
Agroindustries Laboratory, Famaillá Agricultural Experimental Station,
Damiani, Maggi, Gende, Faverin, Eguaras, Marcangeli
National Institute of Agricultural Technology, Tucumán province. The
organoleptic properties assessed were: appearance, consistency,
visible impurities, aroma, flavour and colour. The physicochemical
properties analyzed were: content of water, ash and wax, mechanical
impurities, total resins, total phenols and total flavonoids (expressed
norms (IRAM-INTA 15935-1 Norms, 2008).
For the experiments, a propolis extract in alcohol was prepared.
The propolis powder was dissolved in 70% ethanol at a ratio 1:9 (w/
v) (Cunha et al., 2004). It was then extracted at 60°C for 2 h in
constant shaking, cooled at room temperature and filtered by suction.
After filtration the solution, free of wax and impurities, was dried at
40°C, in order to obtain a soft extract, due to evaporating the alcohol.
The humidity content of the soft extract was determined by drying an
aliquot of this extract to 105ºC, until constant weight (IRAM-INTA
15935-1 Norms, 2008). The relation between wet weight and dry
weight was used to prepare different concentrations of propolis
solutions for the experiments.
As the presence of acaricidal residues in the propolis sample,
remnants of previous treatments, may introduce false results, the
propolis sample used in our experiments was collected from hives
whence the sample was obtained had been managed by alternating
formic acid and thymol applications for at least two year prior to
collecting the propolis.
Biological material
Apis mellifera colonies were used. The hives were placed in an
experimental apiary of the University of Mar del Plata, near Mar del
Plata, Argentina (38º10'06''S; 57º38'10'W). All colonies had been left
untreated for V. detructor for the preceding 12-24 months. Adult
worker bees and capped brood combs were taken from the colonies
Topical and spraying application methods
The soft extract was dissolved in 55% ethanol in order to reduce the
effect of strong ethanol solution on the experimental organisms. The
solutions were prepared considering the humidity content of the soft
extract. The concentrations used in the treatment were 1.25, 2.5, 5,
7.5 and 10% (w/v).
Adult female mites were collected from capped healthy brood by
opening and inspecting individual cells. In order to avoid starvation,
mites were kept on bee larvae or pupae in Petri dishes during the
collection process. Mites that seemed newly moulted, weak or
abnormal were discarded, because they may have different
responses.
For the topical application, methodology adapted from Garedew et
al., (2002) was used. For each treatment, 200 μl of a specified
concentration of propolis were applied to six mites placed on a piece
Propolis toxicity on mites and bees 259

of filter paper (3 x 3 cm) in a Petri dish. Each treatment was Oral administration method
terminated after the allowed contact time (15, 30, 45, 60, 75 and 90 s) For this method, an average of 340.8 ± SE 59 adult worker bees with
by removing the mites from the filter paper, and transferring them no age differentiation, coming from colonies (n = 24) with high V.
into a clean Petri dish (90 x 15 mm). Five replicates for each destructor infestation, were placed in individual cages (16 x 12 x 6 cm)
experimental unit were run. Controls (four replicates) were made by according to Maggi et al., (2010). The bees remained in the cages
treating mites with a 55% ethanol solution for similar contact times. with no food for 4 h and, the propolis solutions were then
All treatments were carried out at room temperature (22-24°C) and administered in 10 ml of 2:1 syrup (sugar diluted in 70% alcohol) as
the treated mites were incubated at 28°C and 60% R.H. Mite activity nutrient. Concentrations tested were: 5, 10, 15 and 20%. Each
was observed under a dissecting microscope at 10 min, 30 min, 60 min treatment was replicated five times. Bees in cages with 10 ml of 2:1
and then after one hour for the next seven hours (total = 10 syrup (as above but without propolis) were included as controls (n =
observation times). Each individual mite was classified as mobile or 4). All cages were maintained at room temperature (22-24ºC and
inactive; it was considered inactive when no leg movement or 65% RH) and a synthetic queen pheromone (Agroindustries
movement of any body part was seen when gently prodded with a Laboratory, INTA Famaillá Agricultural Experimental Station) was
paintbrush. If a mite remained inactive after 8 h from the beginning included. After 24 h all bees were fed only with the 2:1 syrup,
of the treatments, it was considered to be dead. depending on demand. Dead bees and mites were recorded after 24,
The proportion of inactive mites was calculated for each treatment 48 and 72 h. After the experiment was terminated, the bees were
concentration (tested at different contact times) at each observation killed by immersing the cages in 70% alcohol. The final number of
time. In the statistical model, the effects of treatment concentration, mites and bees from each cage was recorded.
contact time (and its interaction) and repetition were included, The proportion of dead mites and bees at each observation period
whereas the observation time was considered as repeated measures was calculated. Arcsine square-root transformed proportions were
in time periods. Least-squares means were compared using Tukey- used. For each observation time, data were analyzed using a model
Kramer test. A 5% level of significance was used, unless otherwise where the dose effects and total number of mites were included as co
stated. Data analysis was conducted using PROC MIXED (SAS -variables for the proportion of dead mites, and the total number of
Institute, 2007). bees as co-variable for the proportion of dead bees. For least-squares
For the spraying application method, Petri dishes (150 x 20 mm) means comparison, the Tukey-Kramer test was utilized with a 5%
padded with absorbent filter paper on the inner bottom and with an level of significance, unless otherwise stated. Data analyses were
extra lid of metallic mesh, were used. Ten adult female mites and ten conducted using PROC MIXED (SAS Institute, 2007).
adult worker bees (free of mites) were placed in every dish. Once the
mites were attached to the body of the bees in each experimental
unit, 3.4 ml of 10% propolis solution were sprayed on the bees
through the metallic lid, using a hand sprayer. The spraying volume
Results
was equivalent to the volume used in the preceding experiment (the Propolis analysis
2
topical method), considering the surface of the filter paper (22 µl/cm , The propolis sample was opaque with shiny irregular fragments of soft
see above). A device with candy and water was placed inside each consistency. Visible impurities were found, especially remains of
unit as food for the bees. Ten bees and ten mites in modified Petri plants, wood and other materials, but vestiges of paint, paper or
dishes sprayed with 55% alcohol were included as controls. Five cardboard were not found. The aroma of the propolis was aromatic
replicates for each experimental group were run. The dishes were resinous; the flavour was sweet and the colour greenish-yellowish
placed in incubators at 28ºC and 70% RH. Death of bees and mites brown. The contents of wax, ash and water of the sample were
was assessed after 24, 48, and 72 h. Mortality was evaluated by 15.06%, 3.65% and 0.82%, respectively. Only 5.89% mechanical
gently prodding each mite with a narrow paintbrush; lack of response impurities were detected. The total amounts of resins, phenols and
to consecutive stimulus over 1 min was considered an indication of flavonoids were 77.45%, 21.74% and 9.18%, respectively.
death. All bees (dead or survivor bees) were visually inspected for the
presence of mites. Topical and spraying methods
The proportion of dead mites and dead bees was calculated for The effects of repetition (F[4,134] = 2.96, P = 0.0220), treatment
each observation time. Data were analysed using GLIMMIX concentration (F[5,134] = 116.62, P < 0.0001), contact time (F[5,134] =
Procedures (SAS Institute, 2007) with logit as the link function. 4.11, P = 0.0017) and observation time (F[9,1467] = 198.46, P < 0.0001)
Statistical significance of pairwise differences between treatments per had a significant effect on mite activity; moreover, a significant
observation time was evaluated using the PDIFF option in conjunction interaction between the effects of treatment concentration and
with the LSMEANS statement. observation time was found (F[45,1467] = 19.28, P < 0.0001; Fig. 1).
260 Damiani, Maggi, Gende, Faverin, Eguaras, Marcangeli

100

90

80

70
Inactive mites (%)

60

50

40

30

20

10

10
7.5
%)

5
n(

2.5
0
a tio

1.25
ntr
nce

Control
10 30 60 120 180 240 300 360 420 480
Observation time (min) Co

Fig. 1. Effect of treatments with different propolis extract concentrations by the topical application method on the activity of V. destructor
during 8 hours of observation. Least-squares means values of inactive mites after treatments (expressed in percentage) are presented here
(n = 174, 10 observation times). The values obtained for the different contact times tested in each treatment concentration were grouped.

100 b
b

90 b
b
80 b
b
b
70 b b b
b
b
60
b
ab
Dead mites (%)

ab
50 ab ab
ab ab

40 ab ab
ab
ab
30

20 a
a a a
a a
ab
10

a a a a a a
0
Control 1.25 2.5 5 7.5 10
-10
Concentration (%)

Fig. 2. Mean percentages of dead mites after treatment during different contact times with diverse propolis extract concentrations by the
topical application method (after 8 hours from the beginning of treatments). Least-squares means + SE values of dead mites are presented
here (n = 174). Means with at least one letter in common do not differ (P > 0.05). Contact times: 15s, 30s, 45s, 60s, 75s, 90s
Propolis toxicity on mites and bees 261

Table 1. Mean percentages of dead mites +Standard Error (SE) and dead bees +SE at 24, 48 and 72 h after treatment of infested bees with
the 10% propolis solution by the spraying application method. *Original analyses were conduced using proportions. Depicted values are back-
transformed least means from the appropriate model. Ten bees and 10 mites per experimental unit (n = 10). Means with different letters
indicate statistically significant differences (P < 0.01) between treatments within groups.

Observation times

24 h 48 h 72 h

Mites Control 2 (2) a 6 (3) a 8 (4) a

Treated 48 (7) b 68 (6) b 78 (6) b

Bees Control 6 (3) a 8 (4) a 10 (4) a

Treated 6 (3) a 8 (4) a 10 (4) a

Table 2. Mean percentage of dead mites +Standard Error (SE) at 24 h, 48 h and 72 h after treatment of naturally infested bees in cages fed
with different concentrations of propolis solution by the oral administration method. *Original analyses were conduced using arcsine square-
root transformed proportions. Depicted values are back-transformed least means from the appropriate model (n = 24).

Propolis Observation times

Concentration 24 h 48 h 72 h

Control 6.35 (4) 6.35(4) 6.35 (4)

5% 1.54 (1) 1.54 (1) 1.54 (1)

10 % 0.00 (0) 0.00 (0) 0.00 (0)

15 % 1.82 (1) 4.32 (3) 4.32 (3)

20 % 4.58 (3) 6.86 (4) 12.97 (7)

Table 3. Mean percentage of dead bees +Standard Error (SE) at 24 h, 48 h and 72 h after treating naturally infested bees in cages with
different concentrations of propolis solutions by the oral administration method. *Original analyses were conduced using arcsine square-root
transformed proportions. Depicted values are back-transformed least means from the appropriate model (n = 24). Means followed by the
same letter are not significantly different (P > 0.05).

Propolis Observation times

Concentration 24 h 48 h 72 h

Control 7.87 (1) ac 9.01 (2) a 9.30 (2) a

5% 4.78 (2) ab 7.71 (3) ab 8.76 (3) ab

10 % 2.45 (0.66) b 2.94 (0.72) b 3.35 (0.86) b

15 % 3.16 (0.36) b 5.75 (0.62) ab 6.60 (0.97) ab

20 % 12.02 (1) c 22.60 (2) c 25.23 (3) c

When the interaction effects between contact time and treatment
concentration, including all observation times, were analyzed, they
were not significant for the effect on V. destructor (F[25,134] = 0.48, P
= 0.9828). When the final observation time (8 h) and a contact time
greater than 30 seconds were considered for the test, the proportions
of dead mites in each treatment concentration were similar (all P > 0.05;
Fig. 2). Eight hours after the beginning of treatment, any mites that
remained inactive were considered dead. From that point on, the
acaricidal effect increased along with increasing concentrations of the
propolis extracts. At the end of the assay, the result of the treatment
with the lowest concentration of propolis extract (1.25%) was not
different from the control (P = 0.9996). However, mites treated
topically with 10% extract showed greater mortality than 60%,
indicating high toxicity even after short contact periods (Fig. 2).
In addition to the mortality effect, the propolis treatment induced
a narcosis effect in V. destructor. This was noticed when the mites
that remained inactive during the firsts hours after treatment
initiation, recovered their activities, regardless of treatment
262
concentration and contact time with the solution. This effect was
more intense with increasing propolis concentration (Fig. 1). In
treatments with higher concentrations (7.5% and 10%) a significant
proportion of mites remained in narcosis during the first two hours
(all P < 0.05), whereas mites exposed to lower treatment
concentrations recovered from narcosis during the first hour post-
treatment (all P < 0.05). However, not all mites recovered from the
narcosis, and those that did not become fully active during the first 8 h biologically active components, such as phenols and flavonoids,
post-treatments were considered dead. This narcotic effect was not
observed in the control group (all P > 0.05; Fig. 1).
The proportion of dead mites differed significantly from the
controls after 24 h (F[1,8] = 13.20, P = 0.0067), 48 h (F[1,8] = 27.52,
P = 0.0008) and 72 h (F[1,8] = 35.41, P = 0.0003) when sprayed with
the 10% propolis solution. In contrast, the proportion of dead bees
after treatment did not differ significantly from the control bees
(F[1,8] = 0, P = 1). Spraying the mites with the 10% propolis extract
thus resulted in 48, 68 and 78% mortality after 24, 48 and 72 h,
respectively (Table 1).
Oral administration method
The prevalence of V. destructor found in the cages was 4.49 ± SE 2
(n = 24). When propolis extracts were administered by the oral
method in cages with infested bees, the proportion of dead mites
after 24, 48 and 72 h was not significant among all concentrations
(F[4,18] = 0.96, P = 0.4516) and the co-variable (F[1,18] = 1.78,
P = 0.1992; Table 2). The effect of the different propolis
concentrations, however, differed significantly for the bees during the
observed period (F[4,18] = 10.87 at 24 h; F[4,18] = 31.95 at 48 h and F
= 23.09 at 72 h; all P < 0.05). The proportion of dead bees at
[4,18]
each observation time was not different within the first three propolis
concentrations administered (all P > 0.05); in the treatment with the
highest concentration this proportion was significantly different from
the control and from the other treatments after 48 and 72 h (P < 0.05; little effect on V. destructor, but the highest propolis concentration
Table 3). caused 25% bee mortality. For this reason, if propolis is to be applied
Discussion
The effect of propolis extracts on several micro-organisms has been
demonstrated (Burdock, 1998) and, when tested against some
parasites, it showed amoebicidal and antigiardial properties, and
affected Trypanosoma cruzi (Higashi and De Castro, 1994; Freitas et
al., 2006; Topalkara et al., 2007). Recent research has shown its
biological effect on causal agents of certain bee pathogens and pests
such as American Foulbrood, caused by Paenibacillus larvae (Gende et geographical zone could be incorporated into honey bee colonies by
al., 2007; Antúnez et al., 2008), the greater wax moth G. mellonella
(Garedew et al., 2004) and V. destructor (Garedew et al., 2002).
The most biologically active components may be obtained when
propolis is extracted in 70% ethanol (Cunha et al., 2004). The major
bioactive compounds are found in the resinous fraction of propolis,
Damiani, Maggi, Gende, Faverin, Eguaras, Marcangeli
and these resins are mainly soluble in alcoholic solutions (Medana et
al., 2008). For this reason, parasites of honey bees are not affected
by the propolis produced in the hive by the bees.
The values of the organoleptic and physicochemical properties in
the propolis sample used in this study were in accordance with data
obtained from other propolis samples from the Pampean region
(Bedascarrasbure et al., 2006). Due to the high concentration of these
propolis collected from that zone has the best quality in Argentina.
With regard to the effect of propolis extracts on V. destructor, a
previous study showed that mites are highly susceptible to propolis
(Garedew et al., 2002). In that research, the propolis in alcoholic
solution was applied topically on mites, the treatment with 10%
solution resulting in 100% mortality, regardless of contact time.
However, in the present study, only 60% of the mites were dead after
a 30 second contact with the 10% propolis solution. These different
results could have been due to a different geographical origin of the
propolis samples, a variable that determines its phenolic fraction
composition. The acaricidal activity of the propolis extracts is probably
due to the presence of bioactive components in this fraction; no
analyses of the propolis sample were made in the previously cited
research. During the present studies mites that had remained in
contact with the propolis showed a narcosis effect, which was more
evident with the higher propolis concentrations. This effect was also
observed by Garedew et al. (2002). When the highest treatment
concentration was sprayed on bees and mites, 48, 68 and 78% of the
mites fell, respectively, 24, 48 and 72 h after the treatment. In
contrast, this treatment was harmless for bees. The narcosis and
lethal effects seen when the mites were maintained with the propolis
solutions clearly indicated the potential of spraying bees with propolis
extracts to control V. destructor.
Feeding infested bees with the propolis extract in sugar syrup had
in hives, either alone or in combination with other natural substances,
it should be used in concentrations under 20% in order to avoid the
death of bees. No data are available about the oral administration of
propolis extract in hives, although in practical beekeeping in Argentina
syrup with unknown concentrations of propolis is commonly added to
feed bees. The propolis may increase honey bee immunity;
enhancement of their defensive response by propolis could also be
important for the control of honey bee diseases (Evans et al., 2006).
The results obtained suggest that propolis extracts from our
spraying, although it is still necessary to adjust the doses and
concentrations to be administered, and the mode of action of the
propolis on mites should be studied. Garedew et al., (2002) suggested
that contact with the propolis solution could lead to a weakening of
the mite cuticle, which would facilitate the entry of active compounds
Propolis toxicity on mites and bees
that are present in propolis. In the present research, oral
administration of propolis had little effect on the mites, even at
concentrations that were toxic for bees. Although propolis, by itself,
may not be useful for mite control, this compound could have an
indirect effect due to stimulating the bees' immune system when
orally applied. Further investigations are required to obtain a better
understanding about the effects of alcoholic propolis extracts on
V. destructor. The variability in the propolis chemical composition,
according to its phytogeographical origin and the various modes of
application in the hives, are the main factors to be considered in
planning future research. Such data would facilitate developing an
integrated management programme for V. destructor that would
reduce the amount of synthetic acaricides applied in the hives.
Acknowledgments
The authors would like to thank Adriana Cano for the assistance in
providing the statistical analysis; and Prof. Gabriela González and an
anonymous reviewer for English grammatical reviews, Miguel Oriental
for providing some laboratory equipment and, Ing. Luis Maldonado,
Ing. Alejandro Álvarez and Tec. Carlos Torne for the analyses of
propolis sample. This research was supported by ANPCyT Project,
PICTO 04 Nº 8-443 to M.E., CONICET and Universidad Nacional de
Mar del Plata.
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